PubMed Health⌕ Search

Biomedical subjects

D Pitt

Publications and source records attributed to D Pitt.

At least 19 recordsLinked to original sources
PubMed2001

Multiple sclerosis: altered glutamate homeostasis in lesions correlates with oligodendrocyte and axonal damage.

Glutamate excitotoxicity, recently demonstrated in an animal model of multiple sclerosis (MS), is evoked by altered glutamate homeostasis. In the present study, we investigated the major regulating factors in glutamate excitotoxicity by immunohistochemistry in MS and control white matter with markers for glutamate production (glutaminase), glutamate transport (GLAST, GLT-1 and EAAT-1), glutamate metabolism (glutamate dehydrogenase [GDH] and glutamine synthetase [GS]), axonal damage (SMI 32) and CNS cell types. Active MS lesions showed high-level glutaminase expression in macrophages and microglia in close proximity to dystrophic axons. Correlation between glutaminase expression and axonal damage was confirmed experimentally in animals. White matter from other inflammatory neurologic diseases displayed glutaminase reactivity, whereas normals and noninflammatory conditions showed none. All three glutamate transporters were expressed robustly, mainly on oligodendrocytes, in normal, control and MS white matter, except for GLT-1, which showed low-level expression around active MS lesions. GS and GDH were present in oligodendrocytes in normal and non-MS white matter but were absent from both active and chronic silent MS lesions, suggesting lasting metabolic impediments. Thus, imbalanced glutamate homeostasis contributes to axonal and oligodendroglial pathology in MS. Manipulation of this imbalance may have therapeutic import.

Adult↗
PubMed2000

Glutamate excitotoxicity--a mechanism for axonal damage and oligodendrocyte death in Multiple Sclerosis?

Glutamate excitotoxicity mediated by the AMPA/kainate-type of glutamate receptors is known not only to damage neurons but also the myelin-producing cell of the central nervous system (CNS), the oligodendrocyte. In Multiple Sclerosis (MS), myelin, oligodendrocytes and axons are lost or damaged as a result of an inflammatory attack on the CNS. Activated immune cells produce glutamate in large quantities by deamidating glutamine via glutaminase. Thus, we hypothesized that during inflammation in MS, glutamate excitotoxicity may contribute to the lesion. This was addressed by treating mice sensitized to develop acute experimental autoimmune encephalomyelitis (EAE) with an AMPA/kainate antagonist, NBQX. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced axonal damage, as indicated by the levels of dephosphorylated neurofilament-H. Despite the clinical differences, NBQX-treatment had no effect on lesion size and did not reduce the degree of CNS inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect at the level of the immune system. In separate studies, infiltrating immune cells present in perivascular cuffs, commonly the site of entry for invading immune cells, were found to express glutaminase in abundance, supporting the production of glutamate in inflammatory lesions. Thus, glutamate excitotoxicity appears to be an important mechanism in autoimmune demyelination and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for MS.

Animals↗
PubMed2000

Insulin-like growth factor-1 fails to enhance central nervous system myelin repair during autoimmune demyelination.

Previous studies have shown that insulin-like growth factor-1 (IGF-1) has beneficial effects, both clinically and histopathologically, on experimental autoimmune encephalomyelitis (EAE), although results vary depending on species and treatment regimen. The present study investigated whether IGF-1, delivered at different time points during the acute and chronic phases of adoptively transferred EAE in SJL mice, had the ability to affect or enhance myelin regeneration. Central nervous system tissue sampled at different stages of treatment was subjected to detailed neuropathological, immunocytochemical and molecular analysis. The results revealed some transient clinical amelioration and low level remyelination after IGF-1 administration during the acute phase of EAE. However, central nervous system tissue from acute phase treated animals sampled at chronic time points and from animals given IGF-1 during the chronic phase revealed no enhancing effect on remyelination in comparison to vehicle-treated controls. Examination of oligodendrocyte progenitor populations also revealed no differences between IGF-1- and vehicle-treated groups. At the cytokine level, the immunomodulatory molecules TGF-beta2 and TGF-beta3 displayed significant decreases that may have contributed to the transient nature of the effect of IGF-1 on EAE. Together with evidence from previous studies, it appears doubtful that IGF-1 is a good candidate for treatment in multiple sclerosis, for which EAE serves as a major model.

Animals↗
PubMed2000

Glutamate excitotoxicity in a model of multiple sclerosis.

Glutamate excitotoxicity mediated by the AMPA/kainate type of glutamate receptors damages not only neurons but also the myelin-producing cell of the central nervous system, the oligodendrocyte. In multiple sclerosis, myelin, oligodendrocytes and some axons are lost as a result of an inflammatory attack on the central nervous system. Because glutamate is released in large quantities by activated immune cells, we expected that during inflammation in MS, glutamate excitotoxicity might contribute to the lesion. We addressed this by using the AMPA/kainate antagonist NBQX to treat mice sensitized for experimental autoimmune encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of multiple sclerosis. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced dephosphorylation of neurofilament H, an indicator of axonal damage. Despite the clinical differences, treatment with NBQX had no effect on lesion size and did not reduce the degree of central nervous system inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect on the immune system. Thus, glutamate excitotoxicity seems to be an important mechanism in autoimmune demyelination, and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for multiple sclerosis.

Animals↗
PubMed2000

Novel Propionibacterium infection in cattle.

OBJECTIVE: To describe four cases of infection in cattle, from geographically different places, with a presumptive new species of Propionibacterium, which causes granulomatous lesions in the head, thorax, abdomen, pelvic area and skin. PROCEDURE: Gross lesions, ranging from 0.5 to 15 cm and detected during routine carcase inspection at the abattoir, were submitted to the laboratory for routine testing in the National Granuloma Submission Program. The bacterium isolated was identified using morphological characteristics, biochemical reactions, cell wall components, products of fermentation and 16S rRNA gene sequencing. RESULTS: Gross lesions submitted for examination consisted of a fibrous outer capsule enclosing thick yellow pus-like material. A Gram-Glynn stain of the histological sections revealed colonies of Gram-positive, filamentous, branching bacteria. Bacteriological culture, cell wall analysis, biochemical reactions and 16S rRNA sequencing identified the organism as a Propionibacterium sp closely related to P cyclohexanicum and the P freudenreichii cluster. CONCLUSION: This is the first report of a Propionibacterium sp closely related to P cyclohexanicum and the P freudenreichii cluster associated with extensive granulomatous lesions in cattle in Queensland. Sequencing data are suggestive of a previously undescribed species of the Propionibacterium genus.

Animals↗
PubMed1999

Neuregulin and erbB receptor expression in normal and diseased human white matter.

Human white matter from non-neurologic cases, multiple sclerosis (MS) and other neurologic diseases (OND, inflammatory and non-inflammatory), was subjected to immunocytochemistry and Western blotting for expression of the neuregulin, glial growth factor-2 (GGF2), and its receptors, erbB2, erbB3 and erbB4. GGF2 has previously been shown to have mitogenic effects upon oligodendrocytes in vitro and an enhancing effect upon remyelination in animals with autoimmune demyelination. In all types of human white matter examined, expression of the ligand GGF2 and its three receptors was consistently found on oligodendrocytes, with higher levels being seen in cases of MS. Expression was also seen, albeit at lower levels, on astrocytes and microglial cells, the latter most commonly in MS and OND. In human lymph node tissue, some lymphocytes were positive for erbB2, erbB3 and erbB4. Western blots confirmed the presence of all three receptors in normal, MS and OND white matter. GGF2 and erbB receptor expression did not correlate with areas of remyelination and reactivity occurred throughout the tissue, with some increase in intensity at the edge of MS lesions. Examination of precursor oligodendrocyte immunoreactivity (with anti-PDGF-Ralpha and NG2), revealed widespread expression throughout both normal and diseased white matter. The presence of GGF2 and its receptors on oligodendrocytes and lymphocytes render this cell type a candidate for functional signaling via this pathway, perhaps in relationship to myelinating activity.

Blotting, Western↗
PubMed1999

Histochemical and ultrastructural characterization of vacuoles and spherosomes as components of the lytic system in hyphae of the fungus Botrytis cinerea.

An integrated approach to acid phosphatase (EC 3.1.3.2) histochemistry by the azo-dye and lead-capture ('Gomori') methods in phosphate-starved hyphae of the fungus Botrytis cinerea revealed strikingly different patterns of localization of activity staining. Reaction product formed with the azo-dye method was found in numerous small organelles (<0.5 microm diameter), which also accumulated the lipophilic dye Nile Red and mislocalized the formazan indicating mitochondrial succinate dehydrogenase activity. Such small organelles were stained only weakly and sporadically with the lead-capture method; instead, lead phosphate deposits were produced mainly in large vacuoles (up to 2.5 microm diam.), similar to those accumulating the vital dye Neutral Red. Additionally, acid phosphatase activity was detected in apical secretory vesicles with the lead-capture method but not with the azo-dye method. Ultrastructural studies by transmission electron microscopy confirmed the presence of large vacuoles which showed evidence of autophagic activity, and of small moderately osmiophilic organelles. The latter are considered to be spherosomes rather than lysosomes because of their weak reaction with the lead-capture method and their high lipid content. It is suggested that their apparently strong reaction with the azo-dye method is caused partly by false localization due to the lipophilic nature of the reaction product.

Acid Phosphatase↗
PubMed1997

Characterization of the egg vesicular components in the seaweed, Fucus serratus L. (Fucales, Phaeophyta), using enzyme histochemistry and vital staining: the search for a lysosome-like body.

Fucus serratus eggs were examined for evidence of the existence of a lysosome-like body using enzyme histochemical and vital staining techniques. Simultaneous coupling azo-dye techniques for lysosomal acid phosphatase proved inappropriate owing to endogenous phenolic binding artefacts. The large number of alginate polysaccharide and polyphenolic egg vesicles interfered with vital staining techniques for lysosomes. Lysosomal esterase activity was detected in the abundant egg lipid bodies. The role of the egg lipid body as an equivalent lysosome-like body of higher plants, the spherosome, is discussed in relation to egg fertilization and early zygote development.

Acid Phosphatase↗
PubMed1996

Ligand-induced regulation of glucagon-like peptide-I receptor function and expression in insulin-secreting beta cells.

Glucagon-like peptide-I (GLP-I) is a potent incretin hormone and mediates its actions via the cyclic AMP (cAMP) pathway. The GLP-I receptor belongs to the family of seven-transmembrane domain receptors coupled to G proteins. We have analyzed the regulation of GLP-I receptor function and expression by its own ligand and the cAMP-dependent pathway in rat insulinoma-derived beta cells (RINm5F). The GLP-I receptor underwent rapid homologous desensitization, which occurred at the receptor level. This was characterized by a reduced binding capacity not mediated by protein kinase A (PKA). GLP-I receptor mRNA levels were down-regulated during incubation of cells by agents increasing cAMP levels including GLP-I itself. This effect was dependent upon time and concentration. Forskolin, the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3, 5-monophosphorothiotate, and GLP-I stabilized the GLP-I receptor mRNA. All induced down-regulation of the GLP-I receptor number within 3 h, a time point at which GLP-I receptor mRNA levels were not decreased. This effect was not influenced by cycloheximide. Therefore, in addition to transcriptional effects, posttranslational mechanisms exist to regulate GLP-I receptor numbers in insulin-secreting cells.

Animals↗
PubMed1995

ACE inhibitor co-therapy in patients with heart failure: rationale for the Randomized Aldactone Evaluation Study (RALES).

Angiotensin converting enzyme (ACE) inhibitor therapy in conjunction with loop diuretics and, possibly, digoxin, is associated with a relatively high incidence of recurrent heart failure and death. Even high doses of ACE inhibitors may not completely suppress the renin-angiotensin-aldosterone system; aldosterone "escape' may occur through non-angiotensin II dependent mechanisms involving corticotropin, atrial natriuretic peptide, serum potassium, and deficient high-density lipoprotein cholesterol concentrations. Addition of spironolactone (an aldosterone receptor blocker) to an ACE inhibitor regimen causes marked diuresis and symptomatic improvement. The Randomized Aldactone Evaluation Study (RALES) was organized to explore the role of combination therapy with spironolactone in patients with heart failure. Patients with New York Heart Association Functional Class II-IV heart failure and left ventricular ejection fractions < or = 40% who were on regimens comprising an ACE inhibitor, loop diuretic, and, possibly, digoxin were randomized to receive placebo or spironolactone in doses of 12.5, 25, 50, or 75 mg per day. Eve at the lowest dose of spironolactone, a significant decrease in plasma N-terminal pro-atrial natriuretic peptide occurred, with concomitant increase in concentrations of plasma renin and urinary aldosterone. As prophylaxis for heart failure, a daily dose of 25 mg of spironolactone and monitoring of serum potassium concentrations are recommended; symptomatic therapy in refractory or severe heart failure may require doses as high as 100 mg b.i.d. The RALES Mortality Trial will follow up 1400 similar patients for 3 years to determine the effect of the addition of spironolactone on combined mortality and hospitalization for heart failure.

Aldosterone↗
PubMed1995

Normal MR appearance of the pituitary gland in the first 2 years of life.

PURPOSE: To investigate changes in the size, shape, and signal intensity of the pituitary gland during the first 2 years of life. METHODS: One hundred consecutive MR studies, spin-echo T1- and T2-weighted sequences, of children 0 to 2 years of age were analyzed. Signal intensity and shape of the gland were graded and its signal intensity measured. RESULTS: There was a statistically significant difference in the signal intensity of both the anterior and posterior pituitary gland and in the shape between children younger than and older than 6 weeks of age and younger than and older than 27 weeks of age. There were definite trends in the size of the gland with age (decrease in height and increase in anteroposterior diameter and width). CONCLUSIONS: There are changes in the pituitary gland in the newborn period that may reflect the ongoing changes in pituitary hormones reported to occur during this time.

Child Development↗
PubMed1993

Calcium homeostasis, signalling and protein phosphorylation during calcium-induced conidiation in Penicillium notatum.

Cytosolic free calcium concentration [Ca2+]c of protoplasts from Penicillium notatum was measured using the permeant acetoxy ester (quin-2-AM) of the calcium-chelating fluorescent dye quin-2. Low uptake of the ester occurred at pH 5.8-7.0 and its subsequent hydrolysis was low. Uptake was promoted by an external pH of 5.0 and significant hydrolysis to quin-2 achieved by adjustment of the internal pH to 7.2, which was near the optimum of the carboxylic esterases responsible for the hydrolysis. Uptake of Ca2+ was biphasic with the average cell calcium concentration of protoplasts increasing from an initial value of 2 mumol to 50 mumol (kg cell water)-1, during attainment of the steady state after 30 min, at which time [Ca2+]c was unchanged at 20 nM but increased to 182 nM at 2-6 h exposure to 2.5 mM-Ca2+. Broadly similar changes in [Ca2+]c were found in protoplasts derived from mycelium samples exposed to Ca2+ over the same period of time. The location of Ca2+ was determined in subfractionated organelles and characterized using enzyme markers and electron microscopy. In 32 h mycelium preloaded with Ca2+ for 6 h, Ca2+ was located principally in the mitochondria with lower concentrations associated with the endoplasmic reticulum, Golgi, vacuoles and plasma membrane components. Calcium was not released by inositol 1,4,5-trisphosphate or the calcium ionophore A23187 from any subcellular fractions obtained from mycelium on Percoll gradients, nor from preparations of vacuoles or plasmalemma vesicles, except in the case of mitochondria where rapid release of the ion was achieved by the addition of 2-5 microM-A23187. The anti-calmodulin agent calmidazolium (R24571) greatly reduced sporulation when addition preceded that of Ca2+. Calcium-induced cultures showed massive novel protein phosphorylation 2 h after addition of the ion which was virtually eliminated by R24571, whilst 1 h and 4-6 h protein phosphorylations, which were also present to some degree in vegetative controls, were substantially reduced. Two-dimensional SDS-PAGE analysis of phosphoproteins confirmed that Ca(2+)-induced mycelium had enhanced capacity for calmodulin-mediated phosphorylation relative to corresponding vegetative cells and that complex differential changes in such phosphorylations occurred during Ca(2+)-induction of the sporulation process.

Calcium↗
PubMed1992

Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.

5'-Nucleotidase↗
PubMed1990

Calcium binding and induction of conidiation in protoplasts of Penicillium cyclopium.

Cell wall-free protoplasts of P. cyclopium could regenerate a cell wall and form mycelia in liquid culture with high rates of viability. When calcium was added to the medium, protoplasts displayed biphasic accumulation with an immediate metabolism-independent adsorption phase, followed by slow metabolism-dependent uptake. Exposure of the protoplasts to Ca2+ for periods of 2 min, followed by incubation in calcium-free medium for 24 hours, was sufficient to induce conidiation with morphogenetic events parallel to those found in cultures containing calcium throughout the incubation period, and similar to those reported in cultures inoculated from conidia. The conidiation event caused by short exposure to calcium could be reversed, within 2 hours of Ca2+ addition, by a brief treatment with the specific calcium chelating agent BAPTA (100 microM), which removed 65 to 75% of the total cell calcium. The results implicate the membrane-bound calcium fraction in the process of conidiation induction.

Calcium↗
PubMed1989

Adenine and pyridine nucleotide levels during calcium-induced conidiation in Penicillium notatum.

Concentrations of adenine and pyridine nucleotides and the associated charge values were examined in extracts of mycelium of Penicillium notatum during vegetative growth and reproductive development promoted by the addition of Ca2+ (10 mmol dm-3). The significant increase in adenylate energy charge promoted by Ca2+ was due to a fall in intracellular AMP and a concomitant rise in ATP concentration. Intracellular concentrations of NADH and NAD fell within 1 h of the addition of Ca2+. The catabolic reduction charge was unchanged by Ca2+ whilst the anabolic reduction charge increased in Ca2+-induced mycelium due to lowered intracellular NADP concentration. Reduced concentration of NADPH in Ca2+-induced mycelium, relative to the vegetative controls, lowered the phosphorylated nucleotide fraction. The results are discussed in relation to metabolic economy during morphogenesis in P. notatum.

Adenine Nucleotides↗